The goal of the proposed research is to define the pathways of hepadnaviral replication leading to persistent infection of hepatocytes. We chose the duck hepatitis B virus (DHBV) for these studies because of the advantages it offers both experimentally and as a simple model for chronic hepatitis B virus infections. Using this experimental system, we can produce genetically altered viral genomes by in vitro mutagenesis of cloned viral DNA. Mutant genomes can be packaged into DHBV particles which can infect cultured hepatocytes. Steps in viral replication can also be studied in a chicken hepatoma cell line which is highly susceptible to transfection with cloned DHBV DNA. Moreover, chronic DHBV infections can be easily established in newly hatched ducklings, which are commercially available. Thus, the DHBV model offers a variety of possibilities for the design of studies to understand the mechanisms of replication and the basis for viral persistence. Using this system, we propose to (i) investigate the role of the preS/S envelope protein of DHBV in regulating replication, virus production, and virulence of DHBV in hepatocytes and ducklings, (ii) determine the basis of the relationship between viral DNA synthesis and nucleocapsid maturation, (iii) define the mechanisms responsible for expression and packaging of pregenomes into nucleocapsids, and (iv) evaluate the importance of extra-cellular virus production in establishing and maintaining of a chronic infection, (v) and determine the lifetime of biologically active cccDNA in vivo and in the absence of DNA replication.